Lymphotactin as an adjuvant

ABSTRACT

Compositions of lymphotactin in cellular immune enhancing amounts may advantageously be administered at very low levels in conjunction with vaccines to provide improved immune response, Suggested dosage such as 1 to 10 ηg in small animals and from 10 μg to 10 mg in large mammals may be administered.

APPLICATION FOR LETTERS PATENT

This application takes priority from Provisional Patent Application60/068,364 filed Dec. 19, 1997.

FIELD OF THE INVENTION

This invention relates to use of lymphotactin as an adjuvant to boostimmune response.

BACKGROUND OF THE INVENTION

The immune response mechanism involves both systemic and localizedmucosal responses to pathogens and to vaccines. The response to theimmunogen or pathogen may be cell-mediated or humoral. (See FundamentalImmunology, 3rd. edition, (W. E. Paul, Editor), Raven Press, NewYork,(1993).) For example, many intestinal pathogens require a mucosalimmune response to provide effective protection from illness.

The use of an adjuvant as a means of enhancing responses to immunogenshas been long known. Adjuvants may function in several ways. Some act onthe immune system to elicit a more effective antibody reaction to theantigen by activating host macrophages, dendritic cells, B cells and Tcells, or enhancing antigen presentation. Adjuvants may enhance immuneresponses by prolonging the release of antigen, increasing antigenuptake, up-regulating antigen processing, stimulating cytokine release,stimulating B cell switching and maturation and/or eliminatingimmuno-suppressor cells. Presently known adjuvants include, aluminumhydroxide and Freund's complete adjuvant. A list of the most effectiveadjuvants would include bacterial toxins which may be administered withthe target immunogen. Sometimes these immune response-enhancingmolecules are bound to the toxin. However, many of these adjuvants causeserious untoward effects.

U.S. Pat. No. 5,571,515 discloses the use of IL-12 as an adjuvant foruse to enhance cell-mediated immunity.

There are four classes of chemokines: CC, C×C, C and C×3 C. Lymphotactinof the C class of chemokines is similar to CC and C×C chemokine familiesthat are common in mammals and that are chemotactic for lymphocytes.

In the adult human, the mucosal surface encompasses more than 300 m² andrequires a significant number of lymphoid cells such as the τδT cellreceptor (τδTCR) intraepithelial lymphocytes (IELs), which producelymphotactin and B cells, which produce secretory immunoglobulin (S-Ig)A antibodies (Abs). S-IgA Abs in the mucosa represent the first line ofdefense against invading pathogens or toxins that, if left unaltered,lead to pathology. Unfortunately, in the context of vaccine development,attempts to induce these protective Abs has not met with great success.

SUMMARY OF THE INVENTION

This invention provides means of enhancing immune response, particularlymucosal immune response, by administration of an immune-enhancingeffective amount of lymphotactin in a pharmaceutically acceptablecarrier. Lymphotactin may be delivered to the mucosa in conjunction withantigen. Mucosal means of application include oral, intranasal, ocular,intravaginal and/or intraurethral administration in liquid orparticulate form.

DETAILED DESCRIPTION OF THE INVENTION

Lymphotactin has been known to be predominately produced by NK and CD8⁺cells as well as in τδT cell receptor (TCR) intraepithelial lymphocytes(IELs). The τδT cells of splenic origin do not produce lymphotactin tothe same degree as similar and more abundant lymphocytes of mucosalorigin, such as those of the upper and lower respiratory,gastrointestinal and reproductive tracts. Lymphotactin used in theexamples was obtained from DNAX Research Institute. Lymphotactinproduced by recombinant technology may be purchased from ResearchDiagnostics, Inc, Flanders, N.J.

Lymphotactin is particularly effective in enhancing immune responses.While bacterial toxins can boost S-IgA, these substrates havedeleterious side effects in humans and other mammals. Fortunately, it isnow possible, by using lymphotactin in accord with the teachings herein,to induce significant and protective Antigen-Specific S-IgA Abs inmucosal secretions. Furthermore, the strategy disclosed herein initiatesserum IgA, IgM and IgG with mixed T helper type 1 and 2 (Th1/Th2)responses. Comparative humoral and cell mediated immune responses havebeen shown to protect laboratory animals against lethal doses of mucosaland systemic pathogens and toxins.

C chemokines such as lymphotactin can be used as a adjuvants in systemicand local, particularly mucosal, vaccine preparations. Theseprotein-based vaccines can facilitate mucosal and systemic immunity toimmunogens whether given in compositions containing in combination the Cchemokine and the target antigen or administered separately to enhanceimmune response to the antigen.

The best known of the effective mucosal vaccines is the Salk poliovaccine. Several antigens are also available to raise immune response tointestinal diseases such as diarrhea arising from E. coli or Shipellaspecies. In all of these and similar instances, the use of lymphotactinto enhance immune response would be appropriate.

Materials and Methods

Immunizations

All mice used were 8 to 10 week old C57BL/6 mice (Charles RiverLaboratories, Willmington, Mass.) housed in laminar cabinets. The micewere free of microbial pathogens, as determined by routine histologicalanalysis. Mice were intranasally immunized with 10 μl (5 μl per nostril)of sterile phosphate buffered saline (PBS), pH 7.5 containing 25 μgchicken egg albumin (OVA from Sigma Chemical Col, St. Louis, Mo.) alone(no lymphotactin) or with 0.01, 0.1, 1.0 or 5 μg of murine lymphotactinon days 0, 7 and 14.

Sample Collection

Serum samples were collected via retro-orbital puncture using sterileheparinized capillary tubes. Vaginal secretion samples were obtained byflushing the vaginal cavity with 50 μl PBS three times for a totalvolume equal to about 150 μl. Fecal pellets were collected, weighed anddissolved in PBS containing 0.1% sodium azide (100 mg fecal pellet per 1ml PBS/sodium azide). These samples were vortexed, centrifuged and thesupernatants were collected for analysis. These mucosal and serumsamples were accumulated at weekly intervals and analyzed for antigen(e.g., OVA)-specific IgA, IgM, IgG, IgE, IgG1, IgG2a, IgG2b and IgG3antibody titers. Mice were sacrificed on day 21 for analysis ofOVA-specific antibody forming cells and T cell proliferative andcytokine profile responses.

Cell Preparation

Submandibular and cervical lymph nodes (SM/CLN), mesenteric lymph nodes(MLN), Peyer's patches (PP), vaginal ileal lymph nodes (ILN) and spleen(SP) suspensions were made by passage of tissue through wire mesh. Afterthe excision of PPs, the small intestine was isolated to determine theIg secreting cells in the intestinal tract which directly relates toprotection against intestinal pathogens. The intestinal tissue was thengently cleaned, minced and treated with 1 mM EDTA in PBS at 37° C. withagitation for 15 to 30 minutes. Next, these tissues were treated withcollagenase in RPMI media for approximately 1 hour. Finally, laminapropria lymphocytes (LPL) were isolated using a percoll (Pharmacia,Uppsala, Sweden) gradient.

The lower respiratory tract (lung) and salivary gland (SG) tissues wereisolated, cleaned, minced and washed in PBS. These tissues were alsodigested with collagenase, isolated and examined to determine theantigen-specific Ig secreting cells and T cell-mediated immunity in thelung and salivary glands, which are important for lower and upperrespiratory immunity.

The nasal tract and nasopharyngeal-associated lymphoid tissue (NALT) wasisolated and passed over sterile glass fiber to acquire a single cellsuspension of lymphocytes. The nasal tract and NALT were studied todetermine the number of Ig secreting cells in the upper respiratorytract needed for protection against respiratory pathogens and toxins.

Antigen-Specific Antibody Titer Detection by ELISA and ELISPOT Assays

Antibody titers in sera and secretions were analyzed by ELISA to confirmthe source of antigen-specific antibodies detected by ELISA,quantitation of vaccine antigen-specific antibody spot forming cellsfrom the SP, PP, MLN, SM/CLN, lung and NALT were enumerated by ELISPOTanalysis.

Enumeration of Antigen-Specific T Cell Proliferative Responses

T cell depleted irradiated (3,000 rads) spleen cells from naive micewere used as feeder cells for T cell proliferation assays. T cells fromthe SM/CLN, MLN, PP, lung, ILN and SP of immunized mice were purifiedusing a nylon wool column. (Purified T cells (2.5×10⁵ cells/ml) werecultured with or without 0.5 mg/ml OVA plus feeder cells (0.5×10⁶cells/ml) in complete RPMI media in round bottom tissue culture treated96well plates. Cells were incubated at 37° in 5% CO₂. After 48 hours ofincubation, 10 μl of 50 μCi/ml [methyl-³ H]-thymidine was added to eachwell. Proliferation or thymidine uptake was measured 18 hours later. Thestimulation index of the various samples was determined and expressed asthe counts per minutes (CPM) of cultures containing OVA divided by theCPMs of cultures lacking OVA.

CD4 T cells that had been isolated using a mouse CD4 isolation columnwere also studied.

RESULTS

The administration of lymphotactin in conjunction with vaccine resultedin increased IgA, IgG, (IgG1, IgG2a, IgG2b, IgG3) and IgM titers in theserum. Increase in fecal IgA and IgG and vaginal IgA and IgG was alsofound. Additionally, augmentation of antigen-specific T cellproliferation of immunized mice was also observed in lymphocytesisolated from SM/CLN, MLN, PP, spleen, lung and ILN. Antibody spotforming cells from NALT, SP, PP, MLN, lung and SM/CLN were shown tosecrete antigen-specific IgA, IgM and IgG antibodies. Hence, it can beseen that the immune responses were increased by exposure of the mucosato lymphotactin.

Compositions of lymphotactin in cellular immune enhancing amounts mayadvantageously be administered at very low levels in conjunction withvaccines. Suggested dosage such as 1 to 10 ηg in small animals and from10 μg to 10 mg in large mammals may be administered. Lymphotactin may beadministered in the usual pharmaceutical carriers such as saline,buffered saline, glucose, etc. Lymphotactin may be administered to themucosa in any manner. Preferred methods of administration involve directapplication to the mucus membranes. Such compositions may, for example,be provided in the form of drops, such as nose, ear or eye drops or insprays. Dry preparations such as lyophilized lymphotactin with powderedcarriers may, for example, be inhaled or sprayed on the mucosa. Suchcompositions may also be provided in capsules or in tablet form foringestion. The lymphotactin may also be administered on a solid supportsuch as a sponge or fiber material. Such administration is particularlyvaluable for use in environments where access to sterile equipment islimited. Compositions for oral ingestion may be enteric coated. Theadjuvants may, additionally, be added to liquids or solids foradministration by mouth. For example, the adjuvants of the invention maybe administered in feed or water or on solid supports such as spongesand fabrics. For example, the adjuvants may be administered in or onbaits.

The adjuvants may be given orally in alkaline solutions containingantigens appropriate for raising antibodies against organisms which giverise to intestinal diseases to raise mucosal antibodies. Alkalinesolutions such as those containing bicarbonates protect antigens andadjuvants from destruction in the upper GI tract.

What is claimed is:
 1. A method of enhancing an immune response byadministering an immune-enhancing effective amount of lymphotactin in apharmaceutically acceptable carrier.
 2. A method of claim 1 wherein thelymphotactin is administered directly to the mucosa.
 3. A method ofclaim 2 wherein the lymphotactin is administered in the form of drops ora spray.
 4. A method of claim 2 wherein the lymphotactin is administeredin powder form.
 5. A method of claim 1 wherein the lymphotactin is on asolid support.
 6. A method of claim 1 wherein the lymphotactin isadministered orally.